Bottom-up proteomics is a useful method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry. Bottom-up proteomics is widely used for qualitative and quantitative characterization of complex biological samples. Given micrograms of material, it is possible to identify more than 10,000 proteins from mammalian cell lysates and over 2,500 proteins from prokaryote lysates. The performance of bottom-up proteomics degrades rapidly for mass-limited samples, such as laser capture microdissected tissues, circulating tumor cells, single embryos, and single somatic cells.
There have been a handful of reports of bottom-up proteomics of nanogram samples using capillary liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). Mann's group identified 2,000 proteins from single pancreatic islets with protein content of several hundred ng (Waanders et al., Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 18902). Karger's group identified 566 proteins from 50 ng of digest of Methanosarcina acetivorans (Yue et al., Anal. Chem. 2007, 79, 938) and 163 proteins from ˜2.5 ng of the tryptic digest of a cervical cancer cell line (Luo et al., Anal. Chem. 2007, 79, 6174). Smith's group detected 870 proteins with an accurate mass and time tags (AMTs)[8] strategy from low nanogram amounts of the digest of Deinococcus radiodurans (Smith et al., Proteomics 2002, 2, 513). Smith's group also reported the detection of the three most abundant proteins in a 0.5 pg sample with the AMTs method, and reported a ˜10 zmole detection limit for one peptide in a bovine serum albumin digest (Shen et al., Anal. Chem. 2004, 76, 144). Dovichi and co-workers used a Q-Exactive mass spectrometer with higher energy collisional dissociation (Olsen et al., Nat. Methods 2007, 4, 709) to identify ˜100 protein groups from 1 ng of a digest of the RAW264.7 macrophage cell line (Sun et al., Rapid Commun. Mass Spectrom. 2013, 27, 157). All of these analyses required at least one hour of instrument time. Thus, faster methods for bottom-up analysis of femtogram amounts of protein digest would be a significant benefit to the proteomics community.
Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has attracted attention for bottom-up proteomics, and this approach consistently outperforms LC-MS/MS for low nanogram samples. The improved performance of CZE for small sample amounts presumably is due to its very simple design, eliminating sample loss on injectors and fittings. Beginning with the pioneering work of Smith's group (Smith et al., Anal. Chem. 1988, 60, 1948), electrospray interfaces have been developed for capillary electrophoresis (Maxwell et al., Anal. Chim. Acta 2008, 627, 25). However, new interfaces are needed to reduce sample dilution due to low sheath flow rates, to eliminate mechanical pumps, to tolerate a wide range of separation buffers, and to stabilize operation in the nanospray regime.